Many vitally important proteins which are secreted (such as immunoglobulins and polypeptide hormones) or present at the cell surface (including cell adhesion molecules and signaling receptors) are produced and folded by the endoplasmic reticulum (ER). If the ER has problems folding these proteins, a compensatory [unreadable]ER stress response[unreadable] (a.k.a. Unfolded Protein Response) is triggered to enhance various ER-related folding processes. ER-produced proteins are frequently N-glycosylated with asparagine-bound sugar polymers (glycans). The glycans are intimately involved in folding of N-glycoproteins, and can also participate in their functions after secretion or reaching the cell surface. The ER-associated lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol provides the glycan (Glc3Man9GlcNAc2) used to make N-glycoproteins. There are 13 human genetic diseases (12 are untreatable), the [unreadable]Congenital Disorders Of Glycosylation[unreadable] (CDG-I) Types Ia-Im, with defective LLO synthesis. Thus, CDG-I cells have ER dysfunction due to poor Nglycosylation, and patients have many clinical difficulties. This research proposal explores the P.I.[unreadable]s discovery that LLO synthesis is one of the processes regulated by the ER stress response, and his hypothesis that the response can be co-opted pharmacologically to repair LLO synthesis abnormalities in CDG-I. Consequently research with this grant has led the P.I. to a new perspective, in which G3M9Gn2-P-P-Dol production is viewed not as a hard-wired process, but rather one which is constantly monitored and adjusted. AIM I will examine the ER stress response as an important regulator of levels of nucleotide-sugars, the biochemical precursors of glycans. AIM II will use CDG-I culture models to test whether pharmacologically-relevant ER stress modulators can counteract LLO synthesis defects, and whether LLO dysfunction in some CDG-I patients[unreadable] cells is corrected by endogenously-triggered ER stress responses. AIMS III and IV will provide important new information about an unexpected activity of mannose-6-phosphate (M6P). The P.I. discovered that M6P is elevated by ER stress, and causes release of the glycan from Glc3Man9GlcNAc2-P-P-dolichol. AIM III will develop custom-synthesized analogues of M6P to elucidate its cellular action. AIM IV will explore the role of M6P-released glycans in ER homeostasis. The clinical relevance of this research is two-fold. First, new strategies for treating glycosylation-deficient human diseases will be evaluated, including some with existing drugs. Second, this work will generate new information about the ER stress response, which governs the productivity of the ER, is essential for the secretory functions of plasma cells (immunoglobulins) and pancreatic islets (insulin), and when aberrantly controlled can cause neurological diseases, cholesterol imbalance, and obesity.